HGS-1 Determination of Fc𝜸R Expression and Binding Specificity on Human Sperm via Fluorescent Detection Methods
Start Date
12-4-2024 9:30 AM
End Date
12-4-2024 11:30 AM
Location
University Readiness Center Greatroom
Document Type
Poster
Abstract
Introduction/Background: There are many sperm surface receptors that play a role in fertilization and the malfunction of these proteins could induce a failure in spermatozoa function, e.g. fertilization potential. Previous research has shown that the interaction between IgG, an immunoglobulin, and Fc gamma receptors (Fc𝜸Rs) may play a role in fertilization and infertility, but none have fully characterized the expression of Fc𝜸Rs on spermatozoa. Fc𝜸R function is mediated by the Fc region of IgG, in which its interaction is critical to produce an effector response. Though Fc𝜸Rs are traditionally thought to govern pro- and anti-inflammatory responses, an understanding of their involvement in fertilization may lead to exciting discoveries and novel therapeutic treatments in reproductive medicine.
Methods: The aim for this project was to identify and distinguish Fc𝜸Rs for IgG on human sperm while utilizing a Fc block to evaluate Fc𝜸R binding specificity for IgG. There are several classes of IgG-binding Fc𝜸Rs, and three were selected for further study: CD16, CD32, and CD64. Two additional receptors for other antibody isotypes were also selected for study: CD23, a receptor for IgE, and FAIM3, an IgM receptor. To identify these receptors on sperm from humans, samples were stained with the designated primary antibody conjugated with a fluorescent tag. Following staining with the primary conjugated antibody, two instruments were used to assess their Fc𝜸R expression. The Nanodrop 3300 detects the overall presence of fluorescence within the sample, while the flow cytometer detects the amount of fluorescent antibody present each individual sperm cell. Two samples were run in this proof-of-concept study.
Results: Via Nanodrop quantification, CD32 was highly expressed in both samples, with greater fluorescence present in Sample B in comparison to Sample A. Flow Cytometry identified the presence of all tested receptors on sperm in Sample A and B, while changes to fluorescent detection was minimal upon adding the Fc block. This shows that the detection of Fc receptors was due to surface expression of each receptor type and was not the result of non-specific binding. Overall, Sample A had a greater mean intensity of expression than Sample B.
Conclusion/Future Directions: Both Nanodrop and flow cytometry methods were reliably able to detect the presence of Fc𝜸Rs and other antibody receptors on the surface of human sperm. Additional data sets will be collected to evaluate their efficacy in assessing receptor expression. Once the expression of specific Fc𝜸Rs and other FcRs are identified on sperm cells, platelet activating factor will be utilized to modulate FcR expression over time, with the potential to impact fertilization success. Future research on these factors will produce a robust understanding of regulated Fc𝜸R expression and male fertility in both humans and other animal species.
HGS-1 Determination of Fc𝜸R Expression and Binding Specificity on Human Sperm via Fluorescent Detection Methods
University Readiness Center Greatroom
Introduction/Background: There are many sperm surface receptors that play a role in fertilization and the malfunction of these proteins could induce a failure in spermatozoa function, e.g. fertilization potential. Previous research has shown that the interaction between IgG, an immunoglobulin, and Fc gamma receptors (Fc𝜸Rs) may play a role in fertilization and infertility, but none have fully characterized the expression of Fc𝜸Rs on spermatozoa. Fc𝜸R function is mediated by the Fc region of IgG, in which its interaction is critical to produce an effector response. Though Fc𝜸Rs are traditionally thought to govern pro- and anti-inflammatory responses, an understanding of their involvement in fertilization may lead to exciting discoveries and novel therapeutic treatments in reproductive medicine.
Methods: The aim for this project was to identify and distinguish Fc𝜸Rs for IgG on human sperm while utilizing a Fc block to evaluate Fc𝜸R binding specificity for IgG. There are several classes of IgG-binding Fc𝜸Rs, and three were selected for further study: CD16, CD32, and CD64. Two additional receptors for other antibody isotypes were also selected for study: CD23, a receptor for IgE, and FAIM3, an IgM receptor. To identify these receptors on sperm from humans, samples were stained with the designated primary antibody conjugated with a fluorescent tag. Following staining with the primary conjugated antibody, two instruments were used to assess their Fc𝜸R expression. The Nanodrop 3300 detects the overall presence of fluorescence within the sample, while the flow cytometer detects the amount of fluorescent antibody present each individual sperm cell. Two samples were run in this proof-of-concept study.
Results: Via Nanodrop quantification, CD32 was highly expressed in both samples, with greater fluorescence present in Sample B in comparison to Sample A. Flow Cytometry identified the presence of all tested receptors on sperm in Sample A and B, while changes to fluorescent detection was minimal upon adding the Fc block. This shows that the detection of Fc receptors was due to surface expression of each receptor type and was not the result of non-specific binding. Overall, Sample A had a greater mean intensity of expression than Sample B.
Conclusion/Future Directions: Both Nanodrop and flow cytometry methods were reliably able to detect the presence of Fc𝜸Rs and other antibody receptors on the surface of human sperm. Additional data sets will be collected to evaluate their efficacy in assessing receptor expression. Once the expression of specific Fc𝜸Rs and other FcRs are identified on sperm cells, platelet activating factor will be utilized to modulate FcR expression over time, with the potential to impact fertilization success. Future research on these factors will produce a robust understanding of regulated Fc𝜸R expression and male fertility in both humans and other animal species.